Journal: Nature

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling

doi: 10.1038/nature14512

Figure Lengend Snippet: a. UV-irradiation and DRB-dependent mobilization of SNRNP40. Quiescent HDFs expressing SNRNP40-GFP were UV-irradiated or DRB treated with doses that inhibit transcription to similar levels. SF mobility was assayed by FRAP. b. Additive effect of combined UV and DRB treatments. FRAP of SNRNP40-GFP in quiescent HDFs treated with DRB, UV, or a combination of both, each at a dose that inhibits RNA synthesis by ≈50%. c. Impaired UV-dependent SF3a1 mobilization in cells lacking ATM activity. SF3a1-GFP mobilization was measured by FRAP in quiescent HDFs derived from an AT patient or a healthy donor. d. ATM-dependent spliceosome mobilization. Quiescent HDFs were treated with 10 μM ATM (KU55933), ATR (VE821) or DNA-PK (NU7441) inhibitors prior to irradiation. GFP-tagged SF3a1 or PRP8 mobility was assayed by FRAP. ATM, but not ATR or DNA-PK inhibition partially prevented the UV-induced SF-mobilization. (a, b, c, d) n=25 , mean ± s.e.m., one-way ANOVA / Bonferroni. e . Reduced UV-induced intron retention in response to ATM silencing. Intron inclusion in RPE cells transfected either with control or ATM silencing siRNAs and subsequently mock-treated or UV irradiated (20 J/m 2 , 6 hrs) was assayed by RT-PCR. f. ATM-dependent changes in intron retention. Intron inclusion was assayed by RT-PCR in untreated, UV irradiated and DRB treated quiescent cells in the presence or absence of 10 μM ATM inhibitor. g. Heatmap of UV-triggered and ATM-dependent transcriptome changes. Quiescent cells were mock-treated or UV-irradiated in the presence or absence of the ATM inhibitor. Transcriptome profiles were generated by RNA-seq. Differentially expressed genes between untreated and UV-irradiated cells (p<0.05) and UV-irradiated cells in the presence or absence of the ATM inhibitor (p<0.05), were clustered in a Heatmap using Pearson correlation. N=1676 differentially expressed transcripts. The observed anti-correlation indicates that UV-inducible transcriptome changes can be, in part, prevented by ATM inhibition. h. Lack of influence of ATM inhibition on DRB-dependent SF mobility. SF mobility was measured by FRAP in untreated or DRB treated HDFs in the presence or absence of 10 μM ATM inhibitor ( n=30 , mean ± s.e.m., one-way ANOVA / Bonferroni).

Article Snippet: Pladienolide B was from Santa Cruz Biotechnology, the ATR inhibitor VE821 from TINIB-Tools, and the ATM inhibitor KU55933 and DNA-PK inhibitor NU7441 from R&D Systems.

Techniques: Irradiation, Expressing, Activity Assay, Derivative Assay, Inhibition, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Generated, RNA Sequencing

Journal: The Journal of cell biology

Article Title: SLX4-XPF mediates DNA damage responses to replication stress induced by DNA-protein interactions.

doi: 10.1083/jcb.202003148

Figure Lengend Snippet: Figure 1. Various DDR proteins are recruited to the LacI-bound lacO array in an S-phase–specific manner. (A) A schematic is shown of the rapid and controlled induction of LacI binding to lacO in U2OS 40–2-6 cells stably expressing HA-ERT2-LacI (ΔNLS). Nuclear accumulation of HA-ERT2-LacI (ΔNLS) was stimulated by treatment with 4-OHT. Representative images are shown of U2OS 40–2-6 ER-LacI cells treated with 4-OHT or vehicle only (EtOH) for 30 min. LacI was detected with anti-LacI immunostain- ing (red) and DNA with DAPI staining (blue). White arrows indicate LacI foci. Scale bar, 10 µm. (B) U2OS 40–2-6 ER-LacI cells were treated with 1 µM 4-OHT or vehicle-only (EtOH) for 80 min and labeled with 10 µM BrdU for the last 20 min. BrdU incorporation was then examined by DNA immunoprecipitation with anti-BrdU antibody (BrdU-IP), followed by quantitative PCR analysis using primer pairs to detect the lacO sequences or LMNB2 replication origin. The relative incorporation of BrdU into the lacO sequences compared with the control region (LMNB2 origin) was calculated. The means ± SD are shown (n = 6). *, P < 0.05 (two-tailed Student’s t test). (C and D) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT for 2 h were double immunostained with the indicated antibodies and counterstained with DAPI. ssDNA was detected with anti-BrdU antibody under nondenaturing conditions. (C) Representative images are shown. Yellow and white arrows indicate colocalization and noncolocalization of DDR proteins with the LacI foci, respectively. Scale bar, 10 µm. (D) Colocali- zation frequencies of the indicated foci with the LacI foci are shown. Values were calculated from the sum scores of at least two independent ex- periments. (E) U2OS 40–2-6 ER-LacI cells were synchronized in G1 phase by lovastatin treatment or in S phase by HU treatment with subsequent release for 4 h (for details, see Materials and methods), then treated with 1 µM 4-OHT for 2 h and double immunostained, followed by DAPI staining and analysis. Colocalization frequencies were calculated from the sum scores of two in- dependent experiments. ***, P < 0.001 (χ2 test). Individual data points from the two experiments are also depicted.

Article Snippet: The following drugs were used: 4-OHT (Abcam), RO-3306 (Sigma), HU (Sigma), lovastatin (LKT Laboratories), VE-821 (Haoyuan Chemexpress), KU-55933 (Sigma), and mirin (Sigma).

Techniques: Binding Assay, Stable Transfection, Expressing, Staining, Labeling, BrdU Incorporation Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Control, Two Tailed Test

Journal: Viruses

Article Title: High Fidelity Deep Sequencing Reveals No Effect of ATM, ATR, and DNA-PK Cellular DNA Damage Response Pathways on Adenovirus Mutation Rate

doi: 10.3390/v11100938

Figure Lengend Snippet: The ability of chemical inhibitors to suppress activation of DNA damage response (DDR) kinases ATM, ATR, and DNA-PK. MRC-5 cells were untreated (Control), treated with the DNA damage inductor NCS, treated with the indicated DDR kinase inhibitor only (ATMi, ATRi or DNA-PKi), or treated with both (neocarzinostatin (NCS) + DDR kinase inhibitor). DDR activation was assayed by fluorescence-based immunocytochemistry using high-content screening to measure fluorescence intensity. The DDR kinase inhibitors used were KU55933 for ATM ( A ), VE-821 for ATR ( B ), and NU7441 for DNA-PK ( C ). Activation of their corresponding targets was determined using antibodies against ATM phosphorylated at Ser1981 ( A ), Chk1 phosphorylated at Ser345 ( B ), or DNA-PK phosphorylated at Ser2056 ( C ). Data are shown as the mean of two independent assays. Fluorescence intensity was measured in 100–300 nuclei foci for each experimental condition. Error bars represent the standard error of the mean.

Article Snippet: ATM inhibitor KU55933 (Sigma-Aldrich, Misuri, USA), ATR inhibitor VE-821 (ApexBio, Houston, USA), and DNA-PK inhibitor NU7441 (ApexBio) were dissolved in DMSO to obtain concentrated stocks.

Techniques: Activation Assay, Control, Fluorescence, Immunocytochemistry, High Content Screening

Journal: Oncogenesis

Article Title: Overexpression of Cyclin E1 or Cdc25A leads to replication stress, mitotic aberrancies, and increased sensitivity to replication checkpoint inhibitors

doi: 10.1038/s41389-020-00270-2

Figure Lengend Snippet: a RPE-1- TP53 wt cells were engineered to overexpress empty, Cyclin E1 or Cdc25A constructs in a doxycycline-inducible manner. Immunoblot shows Cyclin E1, Cdc25A, p53, and Vinculin protein levels at 48 h after addition of doxycycline (dox). b Cells were treated with doxycycline for 48 h, were subsequently labeled for 20 min with CldU (25 µM) and for 20 min with IdU (250 µM). Representative DNA fibers from doxycycline-treated cells are shown. Scale bar measures 10 µm. c Quantification of IdU DNA fiber lengths as described in panel b . At least 266 fibers were analyzed. Graphs show individual data points, median and interquartile range. p -values were calculated using the Mann–Whitney U test . d Examples of chromatin bridges and lagging chromosomes. Cells were stained with α-Tubulin (red) and counterstained with DAPI (blue). Scale bar indicates 10 µm. e Quantification of anaphase and telophase cells containing chromatin bridges and/or lagging chromosomes. The bars represent the mean and standard error or the mean (SEM) from three experiments, n > 25 per experimental condition; p -values were calculated using two-tailed Student’s t -test. f Representative examples of mitotic aberrancies observed in RPE-1 -TP53 wt cells transduced with H2B-EGFP using live-cell microscopy. Scale bar represents 20 µm. g Duration of mitosis as measured by nuclear envelope breakdown to anaphase. Cells were pre-treated with doxycycline for 24 h and subsequently followed with live-cell microscopy using 7 min intervals for the duration of 48 h. p -value was calculated using a Kruskal–Wallis test. h Quantification of aberrant mitoses in cells from panel h . p -values were calculated using absolute values, using Mann–Whitney U test.

Article Snippet: After pretreatment with doxycycline, ATR inhibitor VE-822 (Axon), WEE1 inhibitor MK1775 (Axon MedChem), or Hydroxyurea (Sigma) at the indicated doses, cells were washed in PBS and lysed in MPER lysis buffer (Pierce), complemented with protease and phosphatase inhibitor cocktail (Thermo Scientific).

Techniques: Construct, Western Blot, Labeling, MANN-WHITNEY, Staining, Two Tailed Test, Transduction, Microscopy

Journal: Oncogenesis

Article Title: Overexpression of Cyclin E1 or Cdc25A leads to replication stress, mitotic aberrancies, and increased sensitivity to replication checkpoint inhibitors

doi: 10.1038/s41389-020-00270-2

Figure Lengend Snippet: a Schematic overview of CRISPR/Cas9 gene targeting in TP53 gene. The exon map and protein coding are based on Emsembl entry ENSG00000141510. Placement of the sgRNA sequence is indicated with a horizontal line under exon 4 and the wild type sequence. Sanger sequencing shows that the gRNA targeting exon 4 induced a −7 bp deletion and a +215 bp insertion in RPE- TP53 −/− cl#1 and a −1 deletion and +2 insertion in RPE- TP53 −/− cl#2, leading to frame-shifts in TP53 . b RPE-1- TP53 −/− cl#1 cells were engineered to overexpress empty, Cyclin E1 or Cdc25A constructs in a doxycycline-inducible manner. Immunoblot shows Cyclin E1, Cdc25A, p53, and Vinculin protein levels at 48 h after addition of doxycycline (dox). RPE-1- TP53 wt cells were used as a positive control for p53. c Cells were treated with doxycycline for 48 h, and were then labeled for 20 min with CldU (25 µM) and subsequently for 20 min with IdU (250 µM). Per condition at least 279 fibers were analyzed. Graphs show individual data points, median and interquartile range. p -values were calculated using the Mann–Whitney U test. d Quantification of anaphase or telophase cells containing chromatin bridges or lagging chromosomes. The bars represent mean and standard error or the mean (SEM) from three experiments, n > 25 per experimental condition; p -values were calculated using two-tailed Student’s t -test. e Representative examples of mitotic aberrancies observed in RPE-1 -TP53 −/− cells transduced with H2B-EGFP cells using live-cell microscopy. Scale bar represents 20 µm. f Duration of mitosis as measured by NEB breakdown to anaphase. Cells were pre-treated for 24 h with doxycycline and subsequently followed with live-cell microscopy using 7 min intervals for the duration of 48 h. p -value was calculated using a Kruskal–Wallis test. g Quantification of aberrant mitoses in cells from panel f . p -values were calculated using absolute values, using Mann–Whitney U test.

Article Snippet: After pretreatment with doxycycline, ATR inhibitor VE-822 (Axon), WEE1 inhibitor MK1775 (Axon MedChem), or Hydroxyurea (Sigma) at the indicated doses, cells were washed in PBS and lysed in MPER lysis buffer (Pierce), complemented with protease and phosphatase inhibitor cocktail (Thermo Scientific).

Techniques: CRISPR, Sequencing, Construct, Western Blot, Positive Control, Labeling, MANN-WHITNEY, Two Tailed Test, Transduction, Microscopy

Journal: Oncogenesis

Article Title: Overexpression of Cyclin E1 or Cdc25A leads to replication stress, mitotic aberrancies, and increased sensitivity to replication checkpoint inhibitors

doi: 10.1038/s41389-020-00270-2

Figure Lengend Snippet: a Genome-wide copy number deviation plots of RPE- TP53 −/− cl#1 empty ( n = 47), RPE- TP53 −/− cl#1 -Cyclin E1 ( n = 44) and RPE- TP53 −/− cl#1 - Cdc25A cells ( n = 46). Cells were treated with doxycycline for 120 h. After single cell sorting, genomic DNA was harvested for single-cell whole genome sequencing (sc-WGS). Each panel displays the individual cells in rows, and the chromosomes numbers from 1-X in columns. The modal copy number state is pictured in green, deviations of the modal copy number state, both focal and whole-chromosome, are colored red). b Copy-number alterations (CNAs) per cell were calculated according to the modal state. Medians with interquartile range are depicted and statistical analyses were performed using a One-sided Mann–Whitney U test. c whole numerical chromosomes per cell were counter per single cell. Medians with interquartile range are depicted and statistical analyses were performed using a one-sided Mann–Whitney U test.

Article Snippet: After pretreatment with doxycycline, ATR inhibitor VE-822 (Axon), WEE1 inhibitor MK1775 (Axon MedChem), or Hydroxyurea (Sigma) at the indicated doses, cells were washed in PBS and lysed in MPER lysis buffer (Pierce), complemented with protease and phosphatase inhibitor cocktail (Thermo Scientific).

Techniques: Genome Wide, FACS, Sequencing, MANN-WHITNEY

Journal: Oncogenesis

Article Title: Overexpression of Cyclin E1 or Cdc25A leads to replication stress, mitotic aberrancies, and increased sensitivity to replication checkpoint inhibitors

doi: 10.1038/s41389-020-00270-2

Figure Lengend Snippet: a , b RPE- TP53 wt (panel a ) and RPE- TP53 −/− cl#1 (panel b ) cells were treated with doxycycline for 72 h to induce overexpression of Cyclin E or Cdc25A. Control cells (RPE- TP53 wt ) were then left untreated or were treated with ATR inhibitor (ATRi, VE-822, 1 µM) for 2 h, followed by a 6 h treatment with hydroxyurea (HU, 1 mM) and immunoblotted for ATR-response proteins pATR, pCHK1, pRPA, and γH2AX, and for WEE1-response marker pCDK (Tyr15). Vinculin serves as a loading control. c , d RPE- TP53 wt (panel c ) and RPE- TP53 −/− cl#1 (panel d ) were treated with doxycycline for 72 h to induce overexpression of Cyclin E or Cdc25A. Control cells (RPE- TP53 wt ) were then left untreated or were treated with ATR inhibitor (ATRi, VE-822, 1 µM) for 2 h, followed by a 6 h treatment with hydroxyurea (HU, 1 mM) and immunoblotted for WEE1 response protein pCDK (Tyr15). e RPE-1- TP53 wt cells induced to express Cyclin E1 or Cdc25A were treated with ATR inhibitor (ATRi, VE-822, 0.25 µM) for 8 h as indicated. The percentages of anaphase or telophase cells containing chromatin bridges or lagging chromosomes were quantified. The bars represent mean and standard error or the mean (SEM) from three experiments, n > 25 per condition; p -values were calculated using two-tailed Student’s t -test. f RPE-1- TP53 wt cells induced to express Cyclin E1 or Cdc25A were treated with WEE1 inhibitor (WEE1i, MK-1775, 0.1 µM) for 8 h if indicated. The percentages of anaphase or telophase cells containing chromatin bridges or lagging were quantified. The bars represent mean and SEM from three experiments, n > 25 per experimental condition; p -values were calculated using two-tailed Student’s t -test. g RPE-1- TP53 −/− cl#1 cells induced to express Cyclin E1 or Cdc25A were treated as in panels e and f . The percentages of anaphase or telophase cells containing chromatin bridges or lagging chromosomes were quantified. The bars represent mean and SEM from three experiments, n > 25 per experimental condition; The p -values were calculated by one-way ANOVA ( p < 0.0001) and followed by Sidak’s multiple comparison test. h Percentage of RPE-1- TP53 −/− cl#1 -Cyclin E1-H2B-EGFP cells that showed aberrant mitoses. Cells were pre-treated for 24 h with doxycycline. Cells were then treated with ATR inhibitor (VE-822, 0.25 µM) or WEE1 inhibitor (MK-1775, 0.1 µM), and subsequently followed with live-cell microscopy using 7 min intervals for 48 h. p -values were calculated using a Mann–Whitney U test. i RPE-1- TP53 wt and RPE-1- TP53 −/− cl#1 cell lines were induced to express Cyclin E1 or Cdc25A, and were treated for 3 days with ATR inhibitor (VE-822) in a range from 0 to 3.2 µM, or WEE1 inhibitor (MK-1775) in a range from 0 to 1.28 µM. Subsequently, relative cell survival was assessed using MTT conversion as a proxy. Plots include mean and standard error of the means (SEM) of three biological replicates. Reported p -values were calculated by a Student’s t -test comparing the area under the curve of doxycycline-untreated samples to the curve of the doxycycline-treated samples.

Article Snippet: After pretreatment with doxycycline, ATR inhibitor VE-822 (Axon), WEE1 inhibitor MK1775 (Axon MedChem), or Hydroxyurea (Sigma) at the indicated doses, cells were washed in PBS and lysed in MPER lysis buffer (Pierce), complemented with protease and phosphatase inhibitor cocktail (Thermo Scientific).

Techniques: Over Expression, Control, Marker, Two Tailed Test, Comparison, Microscopy, MANN-WHITNEY

Journal: Oncogenesis

Article Title: Overexpression of Cyclin E1 or Cdc25A leads to replication stress, mitotic aberrancies, and increased sensitivity to replication checkpoint inhibitors

doi: 10.1038/s41389-020-00270-2

Figure Lengend Snippet: a HCC1806 cells transduced with inducible Cyclin E1 construct (sh CCNE1 #1 or sh CCNE1 #2) or control shRNA (sh Luc ) were treated with doxycycline for 2 days, and immunoblotted for Cyclin E1 and β-Actin. Cyclin E1 protein levels were measured and normalized to ‘sh Luc -DOX’ controls for each experiment. Bar graphs reflect the average and standard deviation from eight independent experiments. b Cyclin E1 knock-down after 2 days of doxycycline treatment assessed by immunofluorescence microscopy. The white lines indicate boundaries of nuclei based on DAPI counterstaining. c Average staining intensity of Cyclin E1 as shown in panel b was categorized and plotted in a histogram. The curve fitted is a log-normal Gaussian distribution. At least 450 nuclei were measured. d Percentage of EdU-positive cells after 48 h of doxycycline treatment, measured by flow cytometry. e Representative pictures of clonogenic survival of HCC1806 cells. Cells were plated in six-well plates and allowed to attach for 24 h, after which doxycycline was added. After 14 days, surviving colonies were stained. f , g Colony survival percentages compared to Luc-dox controls f and relative average diameter of colonies counted g in panel f , relative to Luc-dox control. Bars represent the mean and standard error of the mean (SEM) mitotic fraction of two independent experiments. p -values were calculated using two-tailed Student’s t -test h cells were treated with doxycycline for 48 h and sequentially labeled for 20 min with CldU (25 µM) and 20 min with IdU (250 µM). Representative DNA fibers of doxycycline-treated samples are shown. i Quantification of IdU DNA fiber lengths as described in panel h . Per condition, at least 466 fibers were analyzed and corresponding medians with interquartile range are shown. p -value was calculated using Mann–Whitney U test. j γH2AX intensity as measured by flow cytometry in cells treated with and without doxycycline for 48 h. Means and SEM normalized to the untreated luciferase condition are shown from three biological replicates. k Cyclin E1 knock-down was induced by doxycycline treatment for 48 h. Cells were then fixed and the percentage of mitotic aberrancies was quantified. Data represents mean and SEM of three independent experiments; at least 30 mitoses were analyzed for each experimental condition. The p -values were calculated by one-way ANOVA ( p < 0.0001) and followed by Sidak’s multiple comparison test. l Duration of mitosis as measured by NEB breakdown to anaphase. HCC1806 H2B-EGFP cells were pre-treated for 48 h with doxycycline and subsequently followed with live-cell microscopy in 7 min intervals for the duration of 48 h. p -value was calculated using a Kruskal–Wallis test. and subsequently followed with live-cell microscopy using 7 min intervals for 48 h. Duration of mitosis is shown. m Quantification of aberrant mitoses in cells from panel l . p -values were calculated using absolute values, using Mann–Whitney U test.

Article Snippet: After pretreatment with doxycycline, ATR inhibitor VE-822 (Axon), WEE1 inhibitor MK1775 (Axon MedChem), or Hydroxyurea (Sigma) at the indicated doses, cells were washed in PBS and lysed in MPER lysis buffer (Pierce), complemented with protease and phosphatase inhibitor cocktail (Thermo Scientific).

Techniques: Transduction, Construct, Control, shRNA, Standard Deviation, Knockdown, Immunofluorescence, Microscopy, Staining, Flow Cytometry, Two Tailed Test, Labeling, MANN-WHITNEY, Luciferase, Comparison

Journal: Oncogenesis

Article Title: Overexpression of Cyclin E1 or Cdc25A leads to replication stress, mitotic aberrancies, and increased sensitivity to replication checkpoint inhibitors

doi: 10.1038/s41389-020-00270-2

Figure Lengend Snippet: a HCC1806 cell lines were induced to express Cyclin E1 shRNA for 2 days and were then treated with 0.25 µM of ATR inhibitor (ATRi, VE-822) or 0.1 µM of WEE1 inhibitor (WEE1i, MK-1775) for 8 h. Cells were then fixed and stained for DNA content (propidium iodine) and for mitotic population (MPM2) and analyzed using flow cytometry. Bars represent the mean and standard error of the mean (SEM) mitotic fraction of four independent experiments, normalized to untreated Luc-dox; p -values were calculated using two-tailed Student’s t -test b , c HCC1806 cell lines were induced to express Cyclin E1 shRNA and were subsequently treated for 3 days with ATR inhibitor (ATRi, VE-822) (panel b ) or WEE1 inhibitor (WEE1i, MK-1775) (panel c ) in a range from 0 to 1.28 µM. Subsequently, relative cell survival was assessed using MTT conversion as a proxy. Averages and standard error of the means (SEM) of three biological replicates are plotted. Reported p -values were calculated by a Student’s t -test comparing the area under the curve of doxycycline untreated samples to the curve of the doxycycline-treated samples. d Doxycycline-inducible HCC1806 cells were plated in six-well plates and allowed to attach for 24 h. Subsequently, cells were treated with doxycycline and 0.05 µM of ATR inhibitor (ATRi, VE-822) or 0.08 µM of WEE1 inhibitor (WEE1i, MK-1775). After 11 days, surviving colonies were stained. e Quantification of clonogenic survival from panel d . Bars represent the mean and SEM of clonogenic survival, relative to the non-doxycycline treated controls of two independent experiments; p -values were calculated using two-tailed Student’s t -test. f Quantification of colony diameter, relative to non-treated shLuc cells of two independent experiments. Bars represent mean and SEM; p -values were calculated using two-tailed Student’s t -test.

Article Snippet: After pretreatment with doxycycline, ATR inhibitor VE-822 (Axon), WEE1 inhibitor MK1775 (Axon MedChem), or Hydroxyurea (Sigma) at the indicated doses, cells were washed in PBS and lysed in MPER lysis buffer (Pierce), complemented with protease and phosphatase inhibitor cocktail (Thermo Scientific).

Techniques: shRNA, Staining, Flow Cytometry, Two Tailed Test

Journal: The EMBO Journal

Article Title: E2F1 proteolysis via SCF ‐cyclin F underlies synthetic lethality between cyclin F loss and Chk1 inhibition

doi: 10.15252/embj.2018101443

Figure Lengend Snippet: HeLa and CCNF K/O cells were transfected with the indicated siRNA for 48 h were harvested and lysed using SDS. Indicated proteins were resolved by SDS–PAGE and visualised by WB. The image verifies knockdown efficiency of experiments presented in Fig A and B. TFIIH was used as a loading control. WB of indicated proteins as in (A). Ponceau S staining is a loading control. WB of indicated proteins as in (A). Ponceau S staining is a loading control. WB of indicated proteins as in (A). H3 is a loading control. Percentage (%) of γ‐H2AX‐positive cells in HeLa and CCNF K/O after indicated siRNA transfection and 1 μM Chk1i for 20 h. Percent of γ‐H2AX‐positive cells were plotted as mean % ± SD (** P < 0.005). Data were calculated from triplicate experiments using two‐tailed paired t ‐test. Representative FACS plots of γ‐H2AX versus DAPI signal of HeLa cells transfected with empty vector (EV) or HA‐E2F1 untreated or treated with 1 μM Chk1i for 20 h. Cells transfected with non‐targeting siRNA (siNC) or indicated siRNAs were left untreated (DMSO) or treated with ATRi (VE‐821, 1 μM) for 24 h. Cells were harvested and lysed using a Triton X‐100‐based lysis buffer. Indicated proteins were resolved by SDS–PAGE and visualised by WB. Quantification of comet tails in alkaline gel. At least 100 cells, across two slides, were analysed in each condition in three biological replicates. Data are shown as medians, with 25/75% percentile range (box) and 10–90% percentile range (whiskers). P ‐values were calculated using the Mann–Whitney test (two‐tailed). Olive tail moment = (Tail.mean − Head.mean)*Tail%DNA/100 *** P < 0.0005. Cells transfected with indicated siRNAs are subjected to ATR (VE‐821, 1 μM) inhibition for 24 h. Percentage survival of each cell line was assessed via resazurin viability assay and plotted as mean % ± SD (** P < 0.005). Data were calculated from triplicate experiments using two‐tailed paired t ‐test. For simplification, survival rates were normalised to non‐targeting control siNC treated with ATRi.

Article Snippet: VE‐821 ATR inhibitor , Stratech Scientific Ltd , S8007.

Techniques: Transfection, SDS Page, Knockdown, Control, Staining, Two Tailed Test, Plasmid Preparation, Lysis, MANN-WHITNEY, Inhibition, Viability Assay

Journal: The EMBO Journal

Article Title: E2F1 proteolysis via SCF ‐cyclin F underlies synthetic lethality between cyclin F loss and Chk1 inhibition

doi: 10.15252/embj.2018101443

Figure Lengend Snippet:

Article Snippet: VE‐821 ATR inhibitor , Stratech Scientific Ltd , S8007.

Techniques: